PROJECT SUMMARY/ABSTRACT Stroke is the 5th leading cause of death in the United States and the number one leading cause of long-term disability in adults. Thrombolytic agents are available for acute treatment, but their efficacy is modest and their use is limited due to temporal restrictions on administration. Inflammation in stroke is characterized by the activation of glial cells and an influx of peripheral immune cells. Microglia, the resident immune cells in the CNS, play a significant role in the neuroinflammatory response to ischemia. Although microglial activation has typically been considered a pro-inflammatory process, recent publications suggest that microglia could play a protective role in stroke. Progress in the development of effective pharmacotherapies for acute stroke has been limited and centered on neuronal preservation despite extensive and robust data implicating glial cells, including microglia, in the pathophysiology of ischemic injury. Ischemic preconditioning (IPC) is a brief period of ischemia that confers robust neuroprotection against subsequent ischemic events. Elucidating the mechanisms of IPC is considered a critical challenge in stroke research. Recent studies, including from our group, has implicated innate immune pathways, including Toll-like receptors (TLRs) and type 1 interferon (IFN) signaling in IPC mediated protection. In this study we will characterize the potent IPC-like neuroprotective effects of IFN? administration prior to stroke using a variety of methods, including transgenic mice with either systemic IFNAR1 knockout or microglial-specific IFNAR1 knockdown. These studies will help us better understand the role of type 1 IFN signaling in IPC with a focus on microglia specifically. Using immunofluorescent microscopy/in situ hybridization as well as ex vivo flow cytometry techniques, we will also address key mechanistic questions, such as which CNS cell types are producing type I IFNs in response to ischemia, and which cell types are responding to type I IFNs. The overall goal of this project is to characterize the cell type specific contribution of type 1 interferon (IFN) signaling in microglia, infiltrating macrophages, astroctyes, oligodendrocytes and neurons to both IPC- and IFN-induced neuroprotection. Identification of the specific cellular and molecular mechanisms of IPC may hold the key to unlocking a rich source of molecular targets for therapeutic intervention in acute stroke.